As discussed in Bauer et al (Journal of Biotechnology, 2011, in press), in the manufacture of food, there is a demand of stable and well-conditioned starter, protective and probiotic cultures. One of the well established preservation (conservation) processes used during the preparation of these cultures is freeze drying, as it is known to be a gentle drying method leading to minimal damage in micro-organisms. Freeze-drying, also called lyophilisation, is a preservation (or conservation) process whereby the material is frozen (for example into blocks, drops or pellets) at a temperature of below 0° C. The surrounding pressure is then reduced to a range from 10-80 Pa (75-600 mTorr) (Bactéries lactiques De la génétique aux ferments, 2008), generally around 13-27 Pa (100-200 mTorr) and enough heat is added to allow the frozen water in the material to sublime directly from the solid phase to the gas phase. The material can be frozen into the freeze-dryer or introduced directly under a frozen form into the freeze-dryer.
However, freeze drying is a lengthy and energy intensive process (Knorr, 1998; Regier et al., 2004). Moreover, the survival of some bacterial strains is negatively affected by the freezing process (Meryman et al., 1977; Meryman, 2007).
An alternative drying method is vacuum drying, which works at positive temperatures by applying vacuum. Using this drying method at conventional conditions (temperature range between 30 and 80° C.) may cause high losses of cells due to heat damage (Valdramidis et al., 2005). However, heat stresses can be reduced by further reducing the chamber pressure to values just above the triple point of water, which leads to low product temperatures close to 0° C. This process is referred to as Controlled Low-Temperature Vacuum Dehydration (CLTV). King et al. (1989) developed this method for the drying of sensitive food ingredients and also showed that it is applicable to the drying of micro-organisms such as Lactobacillus acidophilus (King and Su, 1993).
Probiotics are well known and are used as dietary supplements. Some probiotics have been preserved by freeze-drying. It is also known that cells which are freeze-dried in the presence of protective agents are better suited to maintain their viability and stability than cells which are freeze-dried without the addition of said protective agents. So generally a protectant is mixed with fresh cell concentrate prior the freeze-drying step.
Freeze-drying can be performed using different techniques. In particular freeze-drying can be performed by tray drying. In this process, the stabilized cell concentrate is loaded directly into freeze dryer trays. The cells are frozen by contact with shelves maintained at a freezing temperature and freeze-dried in a commercial freeze-drier. The resulting cake may then be milled to make a powder, for example which is used in probiotic blends.
Another common freeze-drying process used to preserve cultures is to freeze-dry them in frozen pellet form. The frozen pellets may be formed by dripping stabilized culture onto a chilled surface (such as a chilled barrel or a chilled belt) or into liquid nitrogen. The frozen pellets can be produced and stored independently of freeze-drier availability, and can be easily loaded into freeze drier trays. The resulting dry pellets may then be milled to make a powder, for example which is used in probiotic blends.
Probably the biggest difference between tray-drying and freeze-drying pellets is the rate of freezing. During both freezing processes ice crystals of pure water form, pushing together cells and dissolved solutes into the interstitial spaces between the crystals. When freezing in liquid nitrogen the ice crystals form nearly instantaneously, while freezing in trays allows the ice crystals to grow slowly and hence to a larger size. Freeze-drying removes the ice crystals leaving behind a matrix of interstitial spaces of now dry material. Scanning Electron Microscopy (SEM) of dried material shows that pelletized materials using standard freeze-drying processes have microscopic channels and interstitial matrices and cells are at, or near, the surface. On the contrary tray-dried materials have much larger channels and interstitial matrices and the cells are encapsulated within the matrix material leading to a better protection of the cells. On the other part, standard tray-drying processes are time-consuming in comparison with standard pellets freeze-drying ones, especially due to two factors: a) the slow freezing time limited by heat transfer from the shelves to the material ; b) the slow drying time due to the longer distance needed for water to escape from cells (the resulting cake obtained by the tray-drying process are larger in size than the pellets). Another issue linked with tray drying is the difficulty required by its logistics, e.g. the freeze-drier must be close to the fermentation and the timing of the fermentation harvest and drying must be synchronized.
Therefore there is a need to develop an improved freeze-drying process allowing enhanced characteristics of the micro-organisms (such as a better stability).
The present invention alleviates the problems of the prior art.
In one aspect the present invention provides a process for the preparation of freeze dried micro-organism composition, comprising the step of
(i) subjecting a frozen composition comprising micro-organisms to a drying pressure of from 133 Pa [1000 mT] to 338 Pa [2540 mT] such that at the drying pressure the frozen composition is dried by sublimation of water present in the frozen composition to provide a freeze dried composition comprising the micro-organisms.
In one aspect the present invention provides a process for the preparation of a food or feed, the process comprising                (a) preparing a freeze dried micro-organism by a process comprising the step of                    (i) subjecting a frozen composition comprising micro-organisms to a drying pressure of from 133 Pa [1000 mT] to 338 Pa [2540 mT] such that at the drying pressure the frozen composition is dried by sublimation of water present in the frozen composition to provide a freeze dried composition comprising the micro-organisms;                        (b) combining the freeze dried micro-organism composition with a foodstuff or feedstuff.        
In one aspect the present invention provides a freeze dried micro-organism composition obtainable by a process comprising the step of
(i) subjecting a frozen composition comprising micro-organisms to a drying pressure of from 133 Pa [1000 mT] to 338 Pa [2540 mT] such that at the drying pressure the frozen composition is dried by sublimation of water present in the frozen composition to provide a freeze dried composition comprising the micro-organisms.
In one aspect the present invention provides a freeze dried micro-organism composition prepared by a process comprising the step of
(i) subjecting a frozen composition comprising micro-organisms to a drying pressure of from 133 Pa [1000 mT] to 338 Pa [2540 mT] such that at the drying pressure the frozen composition is dried by sublimation of water present in the frozen composition to provide a freeze dried composition comprising the micro-organisms.
In one aspect the present invention provides a food or feed comprising
(a) a freeze dried micro-organism composition as defined herein; and
(b) a foodstuff or feedstuff.
In one aspect the present invention provides use of drying pressure to prepare a freeze dried micro-organism composition having improved stability and/or improved cell count and/or increased density and/or improved dispersibility,
wherein a drying pressure of from 133 Pa [1000 mT] to 338 Pa [2540 mT] is applied to a frozen composition comprising micro-organisms to dry the frozen composition by sublimation of water present in the frozen composition.
Aspects of the invention are defined in the appended claims.
The present invention provides novel drying techniques for the preparation of freeze dried compositions containing micro-organisms. In particular the present invention provides a process in which frozen compositions containing micro-organisms are freeze-dried.
In this process freeze-drying is performed at pressures which are higher than those normally used for freeze-drying. The skilled man would not have expected to obtain increased micro-organisms characteristics, such as stability, since it would be expected that a high pressure would have been damaging for the micro-organisms.
An advantage of the present invention is that this process (i.e. the use of a high pressure for freeze-drying micro-organisms) may be implemented in different drying techniques such as pellet drying and tray-drying, leading to improved results.
In comparison with the dried pellets obtained using standard freeze-drying techniques (such process using a pressure of 100 mT), cell counts, shelf stability, density and dispersibility of the freeze-dried micro-organisms are enhanced.
In comparison with standard tray-drying process, it has been found that the cell counts and the cell stability of the compositions freeze-dried in accordance with the present invention surpass the ones obtained for commercially tray-dried material. Furthermore the bulk density of freeze-dried compositions in accordance with the present invention after milling is equivalent to the best tray-dried products. Moreover scanning electron microscopy (SEM) images indicate that cells of micro-organisms in the present compositions are encapsulated in a matrix; it is understood that this translates to their better stability.
For ease of reference, these and further aspects of the present invention are now discussed under appropriate section headings. However, the teachings under each section are not necessarily limited to each particular section.